Although much information is now available regarding the extent of antibody variable region diversity and the mechanisms by which it is generated, important quantitative issues remain to be resolved. For example it is known that somatic mutation acts to enhance antibody diversity, but the extent to which antigen-induced variation contributes to the expressed array of variable regions is unclear. Further, the issue of whether the heavy chain switch process is implicated mechanistically in the generation of new heavy chain sequences is still controversial. Finally the question of whether different B cell subpopulations may have distinct variable region repertoires remains to be answered. We propose to examine these questions by conducting an analysis of the diversity of fluorescein-specific antibodies generated both by hybridomas and in splenic fragment cultures. In each of these systems, the diversity of variable region sequences associated with fluorescein-specific antibodies of the IgM, IgGl and IgA heavy chain classes will be measured. Nucleic acid sequencing studies of a number of hybridoma-derived antibodies will be used to complement a less definitive, but more extensive analysis of diversity in short term splenic fragment cultures. The diversity of the response of each of these isotypes will be measured as a function of time following immunization, with particular attention being paid to the relative degrees and types of mutational events occurring in IgM antibodies as opposed to in the other two classes. Finally, using immunodeficient (CBA/NxDBA/2)F1 males and their normal female littermates as the starting model, it is planned to assess the variable region diversity which is expressed by definable B cell subpopulations. Use of fluorescein as the antigen system facilitates the measurement both of antibody affinity and of the fine specificity of receptor sites.